Proteins in the Karyopherinbeta (Kapbeta) family mediate multiple macromolecular import and export pathways between the nucleus and the cytoplasm. The binding of transport substrates and the GTPase Ran to import- Kapbetas is mutually exclusive, while the binding of substrates and Ran to export-Kapbetas is cooperative. This difference in cooperativity is the main biochemical distinction between nuclear import and export, and is likely the key factor in determining nuclear transport directions. A major question in the field concerns mechanistic distinctions between nuclear import and export. It is not known how the homologous import and export-Kapbeta proteins have such disparities in Ran binding activities, cooperativities in Ran and substrate binding, and how they arrive at opposing directions of transport. To address these questions, this proposal describes comparative structural analyses by X-ray crystallography of interactions between import factor Kapbeta2 and export factor CAS with their ligands. The goal of our first aim is to determine the crystal structures of Kaplbeta2 bound to nuclear localization signals (NLSs) of import substrates hnRNP A1 and TAP. These NLSs are non-homologous and are also classified as non-classical NLSs. Structures of their complexes will reveal specificity determinants for Kapbeta2, and inform on the mechanism of substrate/non-classical NLS recognition. Our second aim is to determine the structure of free Kapbeta2. Import by Kapbeta2 begins in the cytoplasm with the unliganded karyopherin. Thus, understanding of the whole import pathway and changes in structure induced by each ligand will require structural studies of the free state as well as of all the complexes. The third and fourth aims will focus on nuclear export. We will determine the crystal structure of the CAS-Kapalpha-RanGppNHp complex. This work will explain for the first time how an export substrate is recognized, it will also will explain the structural basis of positive cooperativity in Ran and export substrate binding by an export-Kapbeta. Finally, we will solve the structure of the Cas-RanGppNHp-RanBP1 complex, a crucial intermediate in the export dissociation process. This work will allow us to understand how export substrate, Kapalpha, is dissociated in the cytoplasm in the last steps of nuclear export. Collectively, these studies will provide insights into specific mechanisms of the different steps in nuclear import by Kapbeta2 and nuclear export by CAS. But more importantly, comparison between these structures of import and export complexes will reveal the mechanistic distinctions between nuclear import and export.